Hydroxyurea (HU) has been shown to increase the proportion of fetal hemoglobin (Hb F) in most sickle cell patients. A low dosage regimen of HU increased total hemoglobin (Hb) levels in some thalassemia intermedia patients by preferentially increasing b-globin biosynthesis. We examined the effect of dosage, duration of exposure, and developmental stage at which HU was given on various hematological parameters and signaling pathways. Erythroid cells in two-phase liquid culture were exposed to HU (5 to 100 mM) either as a pulse for 3 days or continuously for 12 days. Low doses of HU (from 0 to 25 mM) increased Hb levels by up to 2.7-fold, and a high dose of HU (100 mM) increased Hb levels when added at days 3-6 of phase II. No significant changes in Hb levels were observed in response to HU during the late stage of phase II culture (>=9-12 days), although there was a significant dose-dependent increase in Hb F levels. HU exposure during days 0-3 of phase II culture increased the number of erythroid colonies to a maximum of 5-fold at 5 mM HU. GATA-1 mRNA was down-regulated at a high dose of HU and GATA-2 was dose-dependently up-regulated over a lower dosage range. Treatment with 100 mM HU dramatically upregulated the death receptor DR-5, caspase 3, and various other genes related to cell cycle control and apoptosis, as determined by cDNA microarray analysis. In contrast, 10 mM HU modestly up-regulated mRNA levels of the early growth response gene (egr-1). More recently, we used the human erythroleukemia cell line (K562) cells to examine the molecular effects of HU at 4 doses: 0, 10, 100, and 1000 umol/l in 4-day cell culture. Our results confirmed that HU could induce the expression of human gamma-globin gene and there was a dose related decrease in cell growth when K562 cells were incubated with HU. From 8534 screened genes of cDNA microarray analyses with 4-dose-treated K562 cells with HU in two independent experiments, 6580 differentially expressed genes were found, among which 194 genes whose expression was modulated at least 2-fold in K562 cells. Taken together our results indicate that HU exerts concentration-dependent effects on Hb F production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.